Project Summary Molecular motor proteins operate in a complex and crowded cellular environment. Many cargos have opposing motors bound simultaneously; these motors may operate in teams functioning either cooperatively or competitively during organelle transport. High resolution live cell imaging of organelle dynamics suggests that, in addition to vesicular transport, opposing motors drive organelle dynamics including cytoskeletal switching, membrane tethering, and membrane tubulation. Here we will focus on the role of oppositely directed motors at two critical junctures in cellular organelle trafficking: early endosomes and the trans-Golgi network (TGN). We will use in vitro models with increasing complexity to dissect the mechanisms involved, and compare these studies to the dynamics observed in live cells imaged with high temporal and spatial regulation. Specifically, we will focus on the following three aims: (1) To examine the interactions of opposing motors bound to the same cargo, including actin-based and microtubule-based motors, at microtubule-actin filament intersections both in vitro and in the cell. We hypothesize that the tuning of motor parameters determines outcomes at intersections, giving cells an adaptable toolbox to regulate organelle dynamics. We will develop this hypothesis in vitro, starting with myosin-1b and kinesin-1 motors in crossed filament assays and moving to more realistic models of the cellular cytoskeleton using nets of crossed actin filaments and intersecting microtubules. We will develop membrane tubulation assays at cytoskeletal intersections using multiple motors. We will then test the predictions of the model for motor function during tubulation at the TGN in cellular assays. (2) To examine the role of opposing dynein and kinesin-3 motors in modulating membrane tubulation and cargo sorting at early endosomes. We hypothesize that the dynamically balanced forces of opposing dynein and kinesin-3 (KIF16B) motors act at early endosomes to induce tubulation and to facilitate cargo sorting. We will model lipid dynamics induced by the opposing activities of these motors in vitro, and compare these observations to the dynamics of tubulation and sorting of early endosomes in live cell studies. (3) To examine the role of the microtubule-organelle linker protein Hook1 and the BAR-domain protein sorting nexin-6 (SNX6) in the regulation of motor function during endosome tubulation. We hypothesize that the microtubule-organelle linker Hook1 interacts with dynein and dynactin to coordinate the dynamics of sorting and tubulation at the early endosome. SNX6 binds to dynactin and has also been proposed to regulate tubulation at the early endosome. Here, we will examine the roles of Hook1 and SNX6 in regulating tubulation using comparative in vitro reconstitution and live cell imaging approaches. Together, these studies will provide insights into the roles of the three molecular motor families in regulating organelle motility, morphology and remodeling. The guiding hypothesis will be that motors generate asymmetrical forces to move organelles but also to remodel organelle membranes, leading to dynamic sorting and trafficking in the cell.